Effect of n-butanol and cold pretreatment on the cytoskeleton and the ultrastructure of maize microspores when cultured in vitro

The improvement of androgenic induction efficiency of anther cultures is an important goal for plant biotechnology. Although n-butanol has been proven to enhance the androgenic induction, the structural background of this effect has not been investigated in detail. In the present study, the cytological and ultrastructural alterations triggered by two treatments that improve androgenic induction, n-butanol (0.2 % n-butanol for 6 h) and cold pretreatment (7 A degrees C for 10 days) were studied in maize anther cultures. Both treatments increased the frequency of responding microspores, and the highest embryo yield (20.9 embryos per 100 plated anthers compared to 0.5/100 anthers in control) was achieved when a combination of both treatments was applied. To study the effect of the treatments on the cytoskeleton, we labeled microtubules using indirect immunofluorescence and actin filaments by rhodamine phalloidin. Cold pretreatment increased the quantity of actin filaments, whereas the microtubule network remained unaffected. In contrast, n-butanol treatment triggered the reversible depolymerization of microtubules, without having any effect on the actin network. Transmission electron microscopy revealed that n-butanol induced the formation of irregular cell walls. Autophagy-related structures were present during the early development of embryogenic microspores following both treatments, but autophagy was only sustained after fourteen days in microspore-derived structures treated with n-butanol. The results support the concept that the androgenic developmental switch is assisted by cytoskeletal rearrangements, which may facilitate androgenic induction through the promotion of symmetric divisions. The longer duration of autophagic processes may also play a role in the elevated embryo induction after n-butanol treatment.